Electrophoretic methods for spatial analysis

ABSTRACT

The present disclosure provides electrophoretic systems, methods and compositions for spatial analysis, which can serve to magnify or demagnify spatial resolution of analytes of interest that are captured using electrophoresis. Some implementations can use a diverging or converging electric field in an electrophoretic capture system. Such a divergent or convergent electric field, as opposed to a parallel electric field, can be generated by, for example, utilizing different sizes of electrodes associated with or imbedded in substrates. Also provided herein are electrophoretic systems, methods and compositions for spatial analysis, which can serve to selectively migrate one or more analytes from a region of interest in the biological sample for capture using electrophoresis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.17/192,536, filed on Mar. 4, 2021, which claim the benefit of U.S.Patent Application Ser. No. 62/985,103, titled ELECTROPHORETIC METHODSFOR SPATIAL ANALYSIS, filed Mar. 4, 2020, the disclosure of which ishereby incorporated by reference in its entirety.

BACKGROUND

Cells within a tissue have differences in cell morphology and/orfunction due to varied analyte levels (e.g., gene and/or proteinexpression) within the different cells. The specific position of a cellwithin a tissue (e.g., the cell's position relative to neighboring cellsor the cell's position relative to the tissue microenvironment) canaffect, e.g., the cell's morphology, differentiation, fate, viability,proliferation, behavior, signaling, and cross-talk with other cells inthe tissue.

Spatial heterogeneity has been previously studied using techniques thattypically provide data for a handful of analytes in the context ofintact tissue or a portion of a tissue (e.g., tissue section), orprovide significant analyte data from individual, single cells, butfails to provide information regarding the position of the single cellsfrom the originating biological sample (e.g., tissue).

Various methods have been used to prepare a biological sample foranalyzing analyte data in the sample. In some applications, analytes canbe captured from a biological sample, and a high resolution of thecaptured analytes is needed for accurate analysis.

SUMMARY

The present disclosure provides electrophoretic apparatuses, systems,kits, and methods for preparing a biological sample for spatialanalysis.

The spatial resolution of analytes (e.g., nucleic acids or proteins froma biological sample) may be limited due to constraints when patterningcapture areas on an array designed to capture the analytes from abiological sample. The electrophoretic techniques described herein canbe useful in overcoming spatial resolution limitations in a capturearray system.

For example, disclosed techniques can utilize divergent or convergentelectric fields, as opposed to parallel electric fields, as the basisfor an electrophoretic capture array platform to enhance or improvespatial resolution of analytes. Such divergent or convergent electricfields, can be generated by different sizes of electrodes that areassociated with the capture array substrates and/or the samplesubstrates. By way of example, a source electrode can be associated,attached to, or embedded in a sample substrate comprising a biologicalsample and a target electrode can be associated with a capture substratecomprising a capture probe. Alternatively, the sample substrate and thecapture substrate can be made to be conductive respectively so that theycan be used as anode and cathode in an electrophoretic device or system.In some instances, the source electrode can provide an effectiveconductive area different from the target electrode. An electric fieldcan be generated between the source electrode and the target electrode.The ratio of the sizes of the source electrode and the target electrodecan determine a degree of magnification or demagnification of thespatial resolution of analytes being captured. In some instances, thedistance between the source electrode and the target electrode can beadjusted thereby allowing for the resolution to be fine tuned viamagnification or demagnification. In some implementations, the distancebetween the electrodes can be adjusted to reduce or suppress lateraldiffusion of analytes. The analytes can expand or spread out duringmigration from the sample substrate toward the capture substrate throughthe divergent electric field. The analytes captured on the capturesubstrate are considered to be magnified, compared to the analytesoriginally included in the sample on the sample substrate. The analytescontract or are concentrated while migrating from the sample substratetoward the capture substrate through the convergent electric field. Theanalytes captured on the capture substrate are considered to bedemagnified, compared to the analytes originally included in the sampleon the sample substrate.

The techniques described herein can allow for the selection of a regionof interest (ROI) in a sample so that analytes in the ROI are capturedvia magnification or demagnification. Some implementations include anarray of patterned electrodes attached to or imbedded in the sourcesubstrate (e.g., the substrate upon which a sample is located). A subsetof the electrodes in the array can be selected to correspond to a ROI inthe sample and actuated to generate an electric field between theselected subset of electrodes of the source substrate (e.g., comprisinga biological sample) and the target substrate (e.g., the capturesubstrate), so that the analytes in the ROI can be captured viamagnification or demagnification.

Other implementations include a light-actuated conductive film (e.g.,amorphous silicon) located on the source substrate. For example, lightcan be illuminated onto the ROI area of the source substrate comprisinga light actuated conductive film thereby generating an electrophoreticcircuit between the illuminated region on the source substrate and thetarget substrate thereby driving analytes in the ROI from the sourcesubstrate to the target substrate where they can be captured viamagnification or demagnification.

In addition, the techniques described herein provide a solution tofacilitate a targeted analyte capture. For example, some implementationsinclude a mechanism for coupling a photo-thermal effect (e.g., infraredlight illumination) to thermally activate permeabilizing enzymes foundin the same region as the ROI region. This can allow for the separate orsequential capture of analytes in a number of ROIs withoutpermeabilizing the entire sample on the source substrate.

Particular embodiments described herein include an electrophoreticsystem for analyte migration. Such analyte migration can be used forspatial analysis of analytes in a biological sample. In someembodiments, the electrophoretic system includes a first substrate, asecond substrate, a buffer chamber, and a controller. The firstsubstrate may include a first substrate region including a biologicalsample. The first substrate region may be electrically conductive andhave a first surface area. The second substrate may include a secondsubstrate region for receiving analytes from the biological sample. Thesecond substrate region may be electrically conductive and have a secondsurface area. The buffer chamber includes a buffer between the firstsubstrate region and the second substrate region. The controller cangenerate an electric field between the first substrate region and thesecond substrate region such that the analytes are induced to migratefrom the first substrate region (e.g., with a biological sample) towardthe second substrate region (e.g., substrate for receiving analytes).The first surface area of the first substrate region may be larger orsmaller than the second surface area of the second substrate region Insome embodiments, first and second substrate functionalities can bereversed, such that the first substrate could receive analytes while thesecond substrate comprises a biological sample. In such an embodiment,an electric field is generated such that analytes are induced to migratefrom the biological sample on the second substrate toward the firstsubstrate where they are received or captured.

In some implementations, the system can optionally include one or moreof the following features. The second substrate region may includecapture probes for capturing the migrated analytes. In some embodiments,a ratio of the first surface area and the second surface area may rangefrom 0.1 to 100. The first substrate region may include an array ofpatterned conductive regions that are selectively actuated to generatean electric field between the actuated conductive regions and the secondsubstrate region. The first surface area of the first substrate regionmay be smaller than the second surface area of the second substrateregion such that a generated electric field is a divergent electricfield resulting in the analytes on the first substrate region beingmagnified on the second substrate region when captured. The firstsurface area of the first substrate region may be larger than the secondsurface area of the second substrate region such that a generatedelectric field is a convergent electric field resulting in the analyteson the first substrate region being demagnified on the second substrateregion when captured. In some embodiments, the first substrate region isa cathode and the second substrate region is an anode.

The first substrate may be arranged in parallel with the secondsubstrate. The first substrate may be spaced apart at a gap or distancefrom the second substrate. The gap or distance may be selected tomaintain a diffusion rate, that is lower than a threshold diffusionrate, of the analytes migrating under an electric field. The gap ordistance between a first and a second substrate may range from 1 um to10 mm. The first substrate region and the second substrate region mayhave the shape of concentric circular disks, which is shaped to becircular in two dimensions. The first substrate region and the secondsubstrate region may be shaped to be concentrically spherical in threedimensions. The first substrate and the second substrate may be glassslides and each glass slide, or regions thereof, may be coated with aconductive material. The conductive material may include at least one oftin oxide (TO), indium tin oxide (ITO), a transparent conductive oxide(TCO), aluminum doped zinc oxide (AZO), or fluorine doped tin oxide(FTO). The buffer may include a permeabilization reagent. Theelectrophoretic system may further include a light configured toilluminate onto at least a portion of the first substrate region topermeabilize the biological sample on the at least a portion of thefirst substrate region. In addition or alternatively, the sample may bepermeabilized using a detergent before or after enzymatic treatment. Inaddition or alternatively, the sample may be incubated with apermeabilizing agent to facilitate permeabilization of the sample. Inaddition or alternatively, the sample may be permeabilized with a lysisreagent being added to the sample. In addition or alternatively, thesample may be permeabilized by exposing the sample to a protease capableof degrading histone proteins. The electrophoretic system may furtherinclude a power supply and electrical wires connecting the power supplyto the first substrate and the second substrate, or regions thereof.

Particular embodiments described herein include a method for capturinganalytes from a biological sample. The method may include placing thebiological sample on a first substrate region of a first substrate, thebiological sample including analytes; placing capture probes on a secondsubstrate region of a second substrate, the second substrate regionhaving a second surface area different from a first surface area of thefirst substrate region; providing a buffer between the first substrateregion and the second substrate region; and generating an electric fieldbetween the first substrate region and the second substrate region tocause the analytes in the biological sample to migrate from the firstsubstrate region toward the capture probes on the second substrateregion.

In some implementations, the system can optionally include one or moreof the following features. The first substrate region may include anarray of patterned conductive regions. The method may includeidentifying a region of interest (ROI) in the biological sample, andactuating one or more of the patterned conductive regions on the firstsubstrate to generate the electric field, the one or more of thepatterned conductive regions corresponding to the ROI. The first surfacearea of the first substrate region may be smaller than the secondsurface area of the second substrate region such that the electric fieldis configured to be a divergent electric field, and the analytes fromthe ROI in the biological sample are magnified upon capture on thesecond substrate region. The first surface area of the first substrateregion may be larger than the second surface area of the secondsubstrate region such that the electric field is configured to be aconvergent electric field, and the analytes from the ROI in thebiological sample are demagnified upon capture on the second substrateregion. The first substrate may be spaced apart at a distance from thesecond substrate. The distance may be selected to maintain a diffusionrate of the analytes migrating under the electric field. The diffusionrate is lower than a predetermined value.

Particular embodiments described herein include an electrophoreticsystem for capturing an analyte from a biological sample. The system mayinclude a first substrate, a second substrate, a buffer chamber, a lightgenerator, and a controller. The first substrate may include a firstsubstrate region configured to place a biological sample includinganalytes. The biological sample may contain analytes. The firstsubstrate region may include a photoconductive material. The secondsubstrate may include a second substrate region configured to placecapture probes thereon. The second substrate region may be configured tobe conductive. The buffer chamber includes a buffer between the firstsubstrate region and the second substrate region. The light generatormay be configured to emit first light onto a least a portion of thefirst substrate region to permit for the at least a portion of the firstsubstrate region to be electrically conductive. The controller may beconfigured to, based on the first light being emitted onto the at leasta portion of the first substrate region, generate an electric fieldbetween the at least a portion of the first substrate region and thesecond substrate region such that the analytes in the biological samplemigrate from the at least a portion of the first substrate region towardthe capture probes on the second substrate region. The surface area ofthe at least a portion of the first substrate region may be differentfrom a surface area of the second substrate region.

In some implementations, the system can optionally include one or moreof the following features. The surface area of the at least a portion ofthe first substrate region may be smaller than the surface area of thesecond substrate region such that the electric field is configured to bea divergent electric field, and the analytes in the biological sampleare magnified when captured on the second substrate region. The surfacearea of the at least a portion of the first substrate region is largerthan the surface area of the second substrate region such that theelectric field may be configured to be a convergent electric field, andthe analytes in the biological sample are demagnified when captured onthe second substrate region. The first substrate is a glass slide uponwhich is the photoconductive material including amorphous silicon. Thesecond substrate may be a glass slide coated with a conductive material.The conductive material may include at least one of tin oxide (TO),indium tin oxide (ITO), a transparent conductive oxide (TCO), aluminumdoped zinc oxide (AZO), or fluorine doped tin oxide (FTO). The firstlight may include at least one of an ultraviolet (UV) light, visiblelight, or infrared light. The electrophoretic system may include asecond light configured to illuminate onto the at least a portion of thefirst substrate region to permeabilize the biological sample on the atleast a portion of the first substrate region. The second light may beinfrared light. The first substrate region may be configured to be acathode, and the second substrate region may be configured to be ananode. The first substrate may be arranged in parallel with the secondsubstrate. The first substrate may be arranged at an angle to the secondsubstrate. The first substrate may be spaced apart at a distance fromthe second substrate. The distance may be selected to maintain adiffusion rate of the analytes migrating under the electric field to belower than a predetermined value. The distance may range from 1 um to 10cm. The first substrate region and the second substrate region may beshaped to be concentric circular disks. The first substrate region andthe second substrate region may be shaped to be concentricallyspherical. The buffer may include a permeabilization reagent. Theelectrophoretic system may further include a light configured toilluminate onto at least a portion of the first substrate region topermeabilize the biological sample on the at least a portion of thefirst substrate region. In addition or alternatively, the sample may bepermeabilized using a detergent before or after enzymatic treatment. Inaddition or alternatively, the sample may be incubated with apermeabilizing agent to facilitate permeabilization of the sample. Inaddition or alternatively, the sample may be permeabilized with a lysisreagent being added to the sample. In addition or alternatively, thesample may be permeabilized by exposing the sample to a protease capableof degrading histone proteins. The electrophoretic system may include apower supply, and electrical wires connecting the power supply to thefirst substrate region and the second substrate region.

Particular embodiments described herein include a method for migratinganalytes. The method may include placing a biological sample on a firstsubstrate region of a first substrate, the biological sample includinganalytes, and the first substrate region including a photoconductivematerial; placing capture probes on a second substrate region of asecond substrate; providing a buffer between the first substrate regionand the second substrate region; emitting first light onto at least aportion of the first substrate region of the first substrate to permitfor the at least a portion of the first substrate region to beelectrically conductive; and generating an electric field between the atleast a portion of the first substrate region and the second substrateregion to cause the analytes in the biological sample to migrate fromthe at least a portion of the first substrate region toward the captureprobes on the second substrate region. A surface area of the at least aportion of the first substrate region may be different from a surfacearea of the second substrate region.

In some implementations, the system can optionally include one or moreof the following features. The method may further include identifying aregion of interest (ROI) in the biological sample. The ROI may includethe at least a portion of the first substrate region of the firstsubstrate. The method may further include emitting second light onto theat least a portion of the first substrate region to permeabilize thebiological sample on the at least a portion of the first substrateregion.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, patent application, or item ofinformation was specifically and individually indicated to beincorporated by reference. To the extent publications, patents, patentapplications, and items of information incorporated by referencecontradict the disclosure contained in the specification, thespecification is intended to supersede and/or take precedence over anysuch contradictory material.

Where values are described in terms of ranges, it should be understoodthat the description includes the disclosure of all possible sub-rangeswithin such ranges, as well as specific numerical values that fallwithin such ranges irrespective of whether a specific numerical value orspecific sub-range is expressly stated.

The term “each,” when used in reference to a collection of items, isintended to identify an individual item in the collection but does notnecessarily refer to every item in the collection, unless expresslystated otherwise, or unless the context of the usage clearly indicatesotherwise.

Various embodiments of the features of this disclosure are describedherein. However, it should be understood that such embodiments areprovided merely by way of example, and numerous variations, changes, andsubstitutions can occur to those skilled in the art without departingfrom the scope of this disclosure. It should also be understood thatvarious alternatives to the specific embodiments described herein arealso within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the featuresand advantages of this disclosure. These embodiments are not intended tolimit the scope of the appended claims in any manner. Like referencesymbols in the drawings indicate like elements.

FIG. 1 schematically illustrates an example electrophoretic system forpreparing a sample.

FIGS. 2A-B illustrate example magnification and demagnificationtechniques in the electrophoretic system of FIG. 1 .

FIG. 3 is a flowchart of an example process for preparing a biologicalsample.

FIG. 4 schematically illustrates another example electrophoretic systemfor preparing a sample.

FIG. 5 schematically illustrates the electrophoretic system of FIG. 4with a mechanism for providing photo-thermal effect to permeabilize asample.

FIG. 6 is a flowchart of an example process for preparing a biologicalsample.

FIG. 7 illustrates an example configuration of electrodes that can beused in the electrophoretic system.

FIG. 8 is a schematic diagram showing an example of a barcoded captureprobe, as described herein.

DETAILED DESCRIPTION

In general, the techniques described herein relate to electrophoreticcompositions, systems and methods for migrating analytes that may bepresent in a biological sample from one substrate to another. Thetechniques described herein can serve to magnify or demagnify spatialresolution of analytes of interest that are captured using anelectrophoretic system.

Some implementations use a diverging or converging electric field in anelectrophoretic capture system. Such a divergent or convergent electricfield, as opposed to a parallel electric field, can be generated byutilizing different sizes of electrodes associated with or imbedded insubstrates. Alternatively, a divergent or convergent electric field canalso be generated by utilizing different sizes of conductive materialswhich may be found on substrates. Alternatively, a divergent orconvergent electric field can be generated by utilizing different typesof conductive materials found on substrates. In some implementations, asource electrode could be a sample substrate upon which is located abiological sample which may comprise analytes of interest, and a targetelectrode could be a capture substrate wherein capture probes areaffixed (e.g., directly or indirectly, and reversibly ornon-reversibly). The sample substrate and the capture substrate could beconductive for use as an anode and a cathode in electrophoresis. Thesource electrode is configured to provide an effective conductive areadifferent from the target electrode. An electric field can be generatedbetween the source electrode and the target electrode. The ratio of thesizes of the source electrode and the target electrode can determine adegree of magnification or demagnification of the spatial resolution ofanalytes being captured. Further, the distance between the sourceelectrode and the target electrode can be adjusted thereby augmentingthe degree of magnification or demagnification of the resolution. Insome implementations, the distance between the electrodes can beadjusted to reduce or suppress lateral diffusion of analytes. Theanalytes can expand or spread out during migration from the samplesubstrate toward the capture substrate through the divergent electricfield. The analytes captured on the capture substrate are considered tobe magnified, compared to the analytes originally included in the sampleon the sample substrate. The analytes contract or are concentrated whilemigrating from the sample substrate toward the capture substrate throughthe convergent electric field. The analytes captured on the capturesubstrate are considered to be demagnified, compared to the analytesoriginally included in the sample on the sample substrate

The techniques described herein allows for selecting a region ofinterest (ROI) in a biological sample so that the analytes in the ROIcan be captured and either magnified or demagnified. Someimplementations include an array of patterned electrodes from the sourcesubstrate (e.g., the substrate upon which a biological sample islocated). A subset of electrodes in the source substrate can be selectedthat correspond to a ROI in the biological sample, and actuated togenerate an electric field between the selected subset of electrodes ofthe source substrate and the target substrate (e.g., the capturesubstrate), so that the analytes in the ROI can migrate to the targetsubstrate and be captured via magnification or demagnification asdescribed above. For example, the selected subset of electrodes may beactuated by applying a voltage across the selected subset of electrodesto generate the electric field.

Other embodiments include a light-actuated conductive film (e.g.,amorphous silicon) that can be located on the source substrate. In suchan embodiment, if a light-actuated conductive film is on the sourcesubstrate, a light can be used to illuminate an ROI on the sourcesubstrate thereby generating an electrophoretic circuit between theilluminated region on the source substrate and the target substrate sothat the analytes in the ROI can migrate and be captured on the targetsubstrate. In some implementations, amorphous silicon compound can becoated on the substrate using, for example, plasma enhanced chemicalvapor deposition. The photoconductive light can be on the UV and visiblespectrum. Examples of amorphous silicon coating are described further inM. Ristova, et al., Study of hydrogenated amorphous silicon thin filmsas a potential sensor for He—Ne laser light detection, Applied SurfaceScience, vol. 218, Issues 1-4, 30 Sep. 2003, Pages 44-53, the disclosureof which is incorporated herein by reference in its entirety.

In addition, the techniques described herein provide a solution tofacilitate a target analyte capture. For example, some implementationsinclude a mechanism for coupling a photo-thermal effect (e.g., infraredlight illumination) to thermally activate permeabilizing enzymes locallyaround the ROI region. This can allow for separately or sequentiallycapturing analytes in a number of ROIs without permeabilizing the entiresample on the source substrate. For example, water in a liquidpermeabilization reagent (e.g., a buffer comprising a permeabilizationagent such as a protease, e.g.,) can strongly absorb infrared light andbe heated up, which is a photothermal effect. Heating can promote theenzyme activity. For example, protease enzymes (e.g., proteinase K(PROK) enzymes) can become more active at a higher temperature. Inparticular embodiments, the liquid permeabilization agent (e.g., thebuffer comprising a permeabilization agent such as a protease, e.g.,PROK) is kept at a cold temperature (e.g., around 4° C.) to suppressactivity of the permeabilization agent. Local heating via, e.g.,targeted infrared illumination of an ROI can locally activate thepermeabilization agent such as a protease, e.g., PROK, allowing forlocal release of analyte from the ROI for subsequent capture. Forexample, the targeted infrared illumination can elevate the temperatureto, e.g., 20 ° C.-70° C., thereby locally activating thepermeabilization agent, e.g., protease such as PROK. More examples aredescribed in Larsen, et al., Characterization of a recombinantlyexpressed proteinase K-like enzyme from a psychrotrophic Serratia sp.,FEBS Journal, vol. 273, Issue 1, January 2006, pages 47-60 (alsoavailable athttps://febs.onlinelibrary.wiley.com/doi/full/10.1111/j.1742-4658.2005.05044.x),the disclosure of which is incorporated herein by reference in itsentirety,

Referring to FIGS. 1 and 2A-B, an example electrophoretic system 3000 isdescribed. FIG. 1 schematically illustrates an example configuration ofthe electrophoretic system 3000. In some implementations, theelectrophoretic system 3000 can be used to actively cause analytes 3014(e.g., nucleic acids, proteins, charged molecules, etc.) in a biologicalsample 3012 on a source substrate 3002 to migrate to capture probes 3030on a target substrate 3004. The electrophoretic system 3000 can improvethe spatial resolution of captured analytes by actively directinganalytes to diverge FIG. 2A or converge FIG. 25B toward the targetsubstrate so that the analytes are captured on the target substrate inan expanded (e.g., magnified) or condensed (e.g., demagnified) manner.The electrophoretic system 3000 further includes an electrophoreticcontainer 3006, a spacer 3008 between the two substrates, and a controlsystem 3010.

Referring further to FIG. 1 , the first substrate 3002 is configured toreceive a biological sample 3012 that contains one or more analytes3014. By way of example, the biological sample can be one or more cellsor a tissue sample including one or more cells. The first substrate 3002can include a first substrate 3016. In some implementations, the firstsubstrate 3016 can include a patterned array of multiple regions, forexample an array of conductive regions 3018. The biological sample 3012can be prepared on the first substrate 3002 in various ways describedherein.

The first substrate 3002 can be configured as a first electrode in theelectrophoretic system 3000. For example, the first substrate 3002 canbe used as a cathode. In another example, the first substrate 3002 canbe used as an anode.

The first substrate 3002 can be conductive at least in the firstsubstrate region 3016. In some implementations, the first substrate 3002can be configured as a conductive substrate described herein. Forexample, the first substrate 3002 can include one or more conductivematerials that permit for the first substrate 3002 to function as anelectrode. Examples of such a conductive material include, but are notlimited to, tin oxide (TO), indium tin oxide (ITO), a transparentconductive oxide (TCO), aluminum doped zinc oxide (AZO), fluorine dopedtin oxide (FTO), and any combination thereof. Alternatively or inaddition, other materials may be used to provide desired conductivity tothe first substrate 3002. The first substrate 3002 can be coated withthe conductive material. For example, the first substrate 3002 caninclude a conductive coating on the surface thereof, and the sample 3012is provided on the coating of the substrate 3002. By way of example, thefirst substrate can include a glass slide coated with a conductivematerial.

In some embodiments, the first substrate 3002 includes an array ofconductive regions 3018. The array of conductive regions 3018 can bedisposed within the first substrate region 3016. Alternatively, thearray of conductive regions 3018 can be disposed at least partiallyoutside the first substrate region 3016. The conductive regions 3018 canbe patterned in various configurations. In the illustrated example, theconductive regions 3018 are configured in a square pattern.

The conductive regions 3018 can be selectively activated to adjust thesize of the first electrode 3020 in the electrophoretic system 3000. Forexample, a subset of conductive regions 3018 can be selected tocorrespond to a region of interest (ROI) 3022 on the sample 3012, andelectrically activated to generate an electric field between the subsetof conductive regions 3018 and the second substrate 3004. For example,the control system 3010 can be used to receive an input of selecting thesubset of conductive regions 3018 that corresponds to the ROI 3022. Insome implementations, the control system 3010 includes a user interface3064 to receive a user input of the selection. In other implementations,the control system 3010 can automatically select the ROI 3022 or thesubset of conductive regions 3018 corresponding to the ROI 3022.Further, the control system 3010 can be used to electrically activatethe selected subset of conductive regions 3018 by, for example, applyinga voltage across the selected subset of conductive regions 3018, therebygenerating an electric field therebetween. In some implementations, theuser interface 3064 can receive an input of selecting a voltage andother parameters to generate the electric field across the selectedsubset of conductive regions 3018.

Although the first substrate 3002 is illustrated to include a singlesubstrate region 3016 in FIG. 1 , other implementations of the firstsubstrate 3002 can include a plurality of substrate regions configuredto place multiple samples thereon, respectively. Each of such multiplesubstrate regions can be configured similarly to the first substrateregion 3016 described herein.

Referring still to FIG. 1 , the second substrate 3004 comprises captureprobes 3030. In some implementations, the second substrate 3004 incudesa second substrate region 3032. The second substrate 3004 can beconfigured as a second electrode in the electrophoretic system 3000. Forexample, the second substrate 3004 can be used as an anode. In anotherexample, the second substrate 3004 can be used as a cathode. The captureprobes 3030 can be placed on the second substrate region 3032 in avariety of ways described herein. For example, capture probes 3030 canbe directly attached (e.g., reversibly or non-reversibly) to a featureon an array. In another embodiment, capture probes 3030 can beindirectly attached (e.g., reversibly or non-reversibly) to a feature onan array. Alternatively or in addition, the capture probes 3030 can beimmobilized on the second substrate region 3032 of the second substrate3004. Embodiments of the capture probes 3030 are further describedherein, for example with reference to FIG. 8 .

The second substrate 3004 can be conductive in the second substrateregion 3032. In some implementations, the second substrate 3004 can beconfigured as a conductive substrate described herein. For example, thesecond substrate 3004 can include one or more conductive materials thatpermit for the second substrate 3004 to function as an electrode.Examples of such a conductive material include, but are not limited to,tin oxide (TO), indium tin oxide (ITO), a transparent conductive oxide(TCO), aluminum doped zinc oxide (AZO), fluorine doped tin oxide (FTO),and any combination thereof. Alternatively or in addition, othermaterials may be used to provide desired conductivity to the secondsubstrate 3004. In some implementations, the second substrate 3004 canbe coated with the conductive material. For example, the secondsubstrate 3004 can include a conductive coating on the surface thereof,and the capture probes 3030 are provided on the coating of the secondsubstrate 3004. The second substrate can include a glass slide coatedwith a conductive material.

In some implementations, the second substrate 3004 can include an arrayof conductive regions that are selectively actuated, similarly to theconductive regions 3018 of the first substrate 3002. In someimplementations, the second substrate 3004 can have a customizedelectrode pattern to improve capture uniformity.

In some implementations, the first substrate 3002 (or the firstsubstrate region 3016 thereof) and the second substrate 3004 (or thesecond substrate region 3032 thereof) can be be flat and arranged inparallel with each other. For example, the first conductive surface areaof the first substrate 3002 and the second conductive surface area ofthe second substrate 3004 can be flat and arranged in parallel with eachother. Alternatively, at least one of the first substrate 3002 (or thefirst substrate region 3016 thereof, or the first conductive surfacearea thereof) and the second substrate 3004 (or the second substrateregion 3032 thereof, or the second conductive surface area thereof) canbe not flat (e.g., curved or having variations in height or depth). Inaddition or alternatively, the first substrate 3002 (or the firstsubstrate region 3016 thereof, or the first conductive surface areathereof) can be arranged to be angled with the second substrate 3004 (orthe second substrate region 3032 thereof, or the second conductivesurface area thereof). For example, in some embodiments the firstsubstrate and the second substrate can be at a 90° angle one to theother.

In some implementations, the first substrate 3002 (or the firstsubstrate region 3016 thereof, or the first conductive surface areathereof) and the second substrate 3004 (or the second substrate region3032 thereof, or the second conductive surface area thereof) can beshaped as concentric circular or spherical disks. Other configurationsand arrangements are also possible, such as square or rectangular shapesthat are arranged concentrically or non-concentrically.

Referring to FIG. 1 , the first substrate 3002 and the second substrate3004 can be arranged within the electrophoretic container 3006. Theelectrophoretic container 3006 can include a buffer chamber 3050 betweenthe first substrate 3002 and the second substrate 3004. The bufferchamber 3050 is configured to contain a buffer 3052. In someimplementations, the first substrate 3002 and the second substrate 3004can be fully immersed in the buffer 3052. In alternativeimplementations, either or both of the substrates 3002, 3004 can bepartially immersed in the buffer 3052 contained in the electrophoreticcontainer 3006.

The buffer 3052 can be of various types. In some implementations, thebuffer 3052 includes a permeabilization reagent. In some embodiments,the buffer 3052 is contained in the buffer chamber 3050 throughout theelectrophoretic process. The permeabilization reagent can permeabilizethe sample before and/or during electrophoresis. In someimplementations, this chemical permeabilization can be performed inconjunction with light permeabilization such as those described herein,for example with reference to FIG. 5 . In addition or alternatively, thesample can be permeabilized using other methods described herein,independently or in conjunction with permeabilization using thepermeabilization reagent and/or the illumination on the sample.

In some implementations, the buffer can include medium includes at leastone of: a solution including a permeabilization reagent, a solidpermeabilization reagent, and a hydrogel compound including apermeabilization reagent. In some embodiments, the solution includingthe permeabilization agent includes greater than about 2 w/v % sodiumdodecyl sulfate (SDS). In some embodiments, the solution including thepermeabilization agent includes about 8 w/v % to about 12 w/v % SDS. Insome embodiments, the solution including the permeabilization agentincludes proteinase K. In some embodiments, the solution including thepermeabilization agent includes greater than 2 w/v % N-lauroylsarcosineor a sodium salt thereof. In some embodiments, the first member includesan aperture positioned so that when the first substrate is retained, theaperture is aligned with a sample region of the first substrate. In someembodiments, the second member includes at least one aperture positionedso that when the first substrate is retained and the first and secondmembers are aligned by the alignment mechanism, anaperture of the atleast one aperature is aligned with at least a portion of a sampleregion of the first substrate.

In some embodiments, the permeabilization reagent comprises a protease.In some embodiments, the protease is selected from trypsin, pepsin,elastase, or proteinase K.

The biological sample can be permeabilized using permeabilizationreagents and techniques known in the art or otherwise described herein.Biological samples from different sources (e.g., brain, liver, ovaries,kidney, breast, colon, etc.) can require different permeabilizationtreatments. For example, permeabilizing the biological sample (e.g.,using a protease) can facilitate the migration of analytes to thesubstrate surface (e.g., spatially-barcoded features). In someembodiments, the permeabilization reagents can be a detergent (e.g.,saponin, Triton X-100™, Tween-20™). In some embodiments, an organicsolvent (e.g., methanol, acetone) can permeabilize cells of thebiological sample. In some embodiments, an enzyme (e.g., trypsin) canpermeabilize the biological sample. In another embodiment, an enzyme(e.g., collagenase) can permeabilize the biological sample.

In some embodiments the solution can permeabilize the biological sampleand rehydrate the features (e.g., beads) of the shrunken array (e.g.,shrunken hydrogel). In some embodiments, the rehydrating solution canstain the biological sample and rehydrate the features of the shrunkenarray (e.g., beads).

In some embodiments, the rehydrating solution (e.g., permeabilization orstain solution) can diffuse through the biological sample. In someembodiments, the rehydrating solution can reduce diffusion of analytesaway from the substrate. In some embodiments, while diffusing throughthe biological sample, the rehydrating solution can migrate analytestoward the substrate surface and improve the efficiency of analytecapture.

Example structures and processes of using permeabilization reagents arefurther described in PCT Application No. PCT/US19/65100, titled ImagingSystem Hardware, filed Dec. 6, 2019, the disclosure of which isincorporated herein by reference in its entirety.

In some embodiments, a spacer 3008 can be disposed between the firstsubstrate 3002 and the second substrate 3004 to separate the firstsubstrate and the second substrate by a distance D. In some embodiments,the spacer 3008 comprises a non-conductive material, such as plastic,glass, porcelain, rubber, silicone, etc. The distance D can bedetermined to provide a desired level of spatial resolution based onseveral factors, such as, but not limited to, the strength and/orduration of an electric field generated between the first substrate 3002and the second substrate 3004, and other parameters described herein.

The control system 3010 can generate an electric field (-E) between thefirst substrate 3002 and the second substrate 3004. The control system3010 can include a controller 3060 configured to apply a voltage betweenthe first substrate 3002 and the second substrate 3004 using a powersupply 3062. The power supply 3062 can include a high voltage powersupply. The controller 3060 can be electrically connected to the firstsubstrate 3002 and the second substrate 3004, for example usingelectrical wires. The control system 3010 can include a user interface3064 configured to receive a user input of starting or stopping theelectrophoretic process. The user interface 3064 can include varioustypes of input devices, such as a graphic user interface, physical orvirtual buttons, switches, keypads, keyboard, etc., configured toreceive a user input for adjusting operating parameters of the system3000 or for accessing other information (e.g., instructions) associatedwith the system 3000. Examples of operating parameters can include, butare not limited to, voltage applied, duration of voltage application,etc. In some implementations, the input devices can be used to select asubset of conductive regions 3018 that corresponds to a region ofinterest (ROI) 3022 on the sample 3012 so that the subset of conductiveregions are electrically activated to generate an electric field betweenthe subset of conductive regions 3018 and the second substrate 3004. Inaddition, the user interface 3064 can include an output device, such asa display, lamps, etc., configured to output the operating parameters ofthe system 3000 or other information associated with the system 3000.

Referring still to FIG. 1 , in some implementations, the first substrate3002 has a first conductive surface area that can be used as the firstelectrode in the electrophoretic system 3000, and a second substrate3004 has a second conductive surface area that can be used as the secondelectrode in the electrophoretic system 3000. In implementations, wherethe entire first substrate region 3016 is configured and used for thefirst electrode, the surface area of the first substrate region 3016 canbe the first conductive surface area. Similarly, where the entire secondsubstrate region 3032 is configured and used for the second electrode,the surface area of the second substrate region 3032 can be the secondconductive surface area. Alternatively, where a portion of the firstsubstrate region 3016 is configured and used for the first electrode,the surface area of the portion of the first substrate region 3016 canbe the first conductive surface area. For example, where the firstsubstrate region 3016 includes the array of conductive regions 3018, thesubset of conductive regions 3018 that is activated can be the firstconductive surface area. Similarly, where a portion of the secondsubstrate region 3032 is configured and used for the second electrode,the surface area of the portion of the second substrate region 3032 canbe the second conductive surface area.

In some implementations, the first conductive surface area of the firstsubstrate 3002 is different from the second conductive surface area ofthe second substrate 3004, so that a divergent or convergent electricfield is generated between the first and second conductive surface areasin the electrophoretic system 3000.

As illustrated in FIGS. 26 and 27A, a divergent electric field 3040 canbe generated when the first conductive surface area of the firstsubstrate 3002 is smaller than the second conductive surface area of thesecond substrate 3004. In the divergent electric field 3040, theanalytes 3014 can migrate from the sample 3012 on the first substrate3002 toward the second substrate 3004 in a diverging manner. Forexample, the analytes 3014 expand or spread out during migration throughthe divergent electric field 3040 prior to being captured on the secondsubstrate 3004. In this case, the analytes 3014 captured on the secondsubstrate 3004 are considered to be magnified, compared to the analytesoriginally included in the sample 3012 on the first substrate 3002.

As illustrated in FIG. 2B, a convergent electric field 3042 can begenerated when the first conductive surface area of the first substrate3002 is larger than the second conductive surface area of the secondsubstrate 3004. In the convergent electric field 3042, the analytes 3014can migrate from the sample 3012 on the first substrate 3002 toward thesecond substrate 3004 in a converging manner. For example, the analytes3014 contract or are concentrated while migrating through the convergentelectric field 3042 and captured on the second substrate 3004. In thiscase, the analytes 3014 captured on the second substrate 3004 areconsidered to be demagnified, compared to the analytes originallyincluded in the sample 3012 on the first substrate 3002.

Magnification or demagnification can be adjusted and controlled by usingdifferent sizes of the first electrode of the first substrate 3002 andthe second electrode of the second substrate 3004. For example,magnification or demagnification can be adjusting based on a ratio ofthe first conductive surface area of the first substrate 3002 over thesecond conductive surface area of the second substrate 3004. By way ofexample, in implementations where the first and second conductivesurface areas are circular, a magnification factor M can be approximatedas:

$M \sim \frac{{Radius}{of}{Second}{Conductive}{Surface}{Area}}{{Radius}{of}{First}{Conductive}{Surface}{Area}}$

In some implementations, the ratio between the first conductive surfacearea and the second conductive surface area can range between 0.1 and100. In other implementations, the ratio between the first conductivesurface area and the second conductive surface area can range between0.01 and 1000. Other ratios are also possible.

By way of example, as illustrated in FIG. 2A, the magnification can beincreased by providing a larger second conductive surface area of thesecond substrate 3004 than the second conductive surface area of thesecond substrate 3004 in FIG. 1 . As illustrated in FIG. 2B, ademagnification can occur when the first conductive surface area of thefirst substrate 3002 is larger than the second conductive surface areaof the second substrate 3004.

In addition or alternatively, the distance D between the first substrate3002 and the second substrate 3004 can be controlled to adjust themagnification or demagnification. Further, the distance D can becontrolled to suppress potential low resolution capture of the analytesthat may result from lateral diffusion. For example, the distance D canbe selected to maintain a diffusion rate of the analytes migrating undera divergent or convergent electric field to be lower than apredetermined value. In some implementations, the distance D can rangefrom 1 um to 10 mm. In other implementations, the distance D can rangefrom 0.1 um to 10 mm.

FIG. 3 is an exemplary flowchart of a process 3100 for capturinganalytes from a biological sample on a sample substrate to a capturesubstrate using electrophoresis. In some implementations, the process3100 can be performed using the electrophoretic system 3000 describedwith reference to FIGS. 1 and 2A-B. The process 3100 can include placinga sample on a first substrate (3102) and placing a capture probe on asecond substrate (3104). The sample can be placed on the first substratein various ways described herein. The capture probes can be attached tothe second substrate in various ways described herein. The first andsecond substrates can be configured and used as electrophoreticelectrodes. In some implementations, the first and second substrates canbe configured as conductive substrates as described herein, such as byincluding a conductive material in the substrates or providing aconductive coating on an upper or lower surface of the substrates.

As described herein, a conductive surface area of the first substrate isdifferent from a conductive surface area of the second substrate. Wherethe conductive surface area of the first substrate is smaller than theconductive surface area of the second substrate, a divergent electricfield is generated between the first and second substrates, and theanalytes can be captured on the second substrate in a magnified way.Where the conductive surface area of the first substrate is larger thanthe conductive surface area of the second substrate, a convergentelectric field is generated between the first and second substrates, theanalytes can be captured on the second substrate in a demagnified way.

The process 3100 can include providing a buffer between the firstsubstrate and the second substrate (3106). In some implementations, theprocess 3100 can include arranging a spacer between the first and secondsubstrates so that the first substrate is arranged at a distance fromthe second substrate. The distance can be selected to maintain adiffusion rate of the analytes migrating under the electric field to belower than a predetermined value. As described herein, the spacer can bemade of a non-conductive material and used to provide a buffer chamberbetween the first and second electrodes. The buffer can be contained ina buffer chamber that is provided by the spacer and used to at leastpartially immerse the first substrate, the second substrate, or both. Insome implementations, the buffer can include a permeabilization reagent.The permeabilization reagent can permeabilize the sample before and/orduring electrophoresis. In some implementations, this chemicalpermeabilization can be performed in conjunction with lightpermeabilization such as those described herein, for example withreference to FIG. 5 . In addition or alternatively, the sample can bepermeabilized using other methods described herein, independently or inconjunction with permeabilization using the premeabilization reagentand/or the illumination on the sample. Other buffers as described hereincan be used in other implementations.

The exemplary process 3100 can include identifying a region of interest(ROI) on the first substrate (3108). The ROI can be selected to target asubset of the sample that is of particular interest and capture theanalytes therefrom. The process 3100 can include generating an electricfield (e.g., a divergent or convergent electric field) between the ROIof the first substrate and the second substrate (3110). For example, thefirst substrate can be selectively activated such that only the regionof the first substrate corresponding to the ROI can be used as anelectrode during electrophoresis. The electric field generated betweenthe ROI of the first substrate and the second substrate can causeanalytes to migrate from the ROI of the first substrate toward thesecond substrate in a diverging or converging manner depending on thetype of the electric field (e.g., either a divergent electric field or aconvergent electric field). In some implementations, the first substrateincludes an array of patterned conductive regions that are selectivelyactivated to correspond to the ROI of the first substrate.

Referring to FIG. 4 , another exemplary electrophoretic system 3200 isdescribed. In some implementations, the electrophoretic system 3200 canbe used to actively cause analytes (e.g., nucleic acids, proteins,charged molecules, etc.) in the sample on a source substrate to migrateto capture probes on a target substrate. The electrophoretic system 3200can improve spatial resolution of captured analytes by activelydirecting analytes to diverge or converge toward the target substrate sothat the analytes are captured on the target substrate in an expanded(e.g., magnified) or condensed (e.g., demagnified) manner. Theelectrophoretic system 3200 can use a light-activated conductivematerial included in at least one of the source substrate and the targetsubstrate to select a region of interest on the sample, and/or adjust adegree of magnification or demagnification of the analytes captures onthe target substrate. In some implementations, the electrophoreticsystem 3200 includes a first substrate 3202, a second substrate 3204, anelectrophoretic container 3206, a spacer 3208, and a control system3210.

Referring still to FIG. 4 , the first substrate 3202 is configured toreceive a sample 3212 that contains analytes 3214. The sample 3212includes a biological sample, such as a cell or a tissue sectionincluding a cell. The first substrate 3202 can include a first substrateregion 3216 for receiving the sample 3212 thereon. The sample 3212 canbe prepared on the first substrate 3202 in various ways describedherein. The first substrate 3202 is configured to be used as a firstelectrode in the electrophoretic system 3200. For example, the firstsubstrate 3202 can be used as a cathode. In another example, the firstsubstrate 3202 can be used as an anode.

The first substrate 3202 can be conductive at least in the firstsubstrate region 3216. In some implementations, the first substrate 3202can be configured as a conductive substrate described herein. Forexample, the first substrate 3202 can include a light-activatedconductive material 3218 that is activated to be electrically conductivewhen exposed to light of predetermined characteristics (e.g.,wavelength). An example of such a light-activated conductive materialincludes amorphous silicon. See, e.g., Valley, J K et al.“Optoelectronic tweezers as a tool for parallel single-cell manipulationand stimulation.” IEEE transactions on biomedical circuits and systemsvol. 3,6 (2009): 424-31. doi:10.1109/TBCAS.2009.2031329, which is herebyincorporated by reference in its entirety. Other materials may be usedto provide such selective conductivity to the first substrate 3202. Thelight-activated conductive material 3218 can be coated to be a film orlayer on the surface of the first substrate 3202 (e.g., a glass slide),and the sample 3212 is provided on the coating of the substrate 3202.

The light-activated conductive material 3218 on the first substrate 3202can be selectively activated to adjust the size of the first electrode3220 in the electrophoretic system 3200. The light-activated conductivematerial 3218 can be used to create a free form of customized region ofinterest (ROI) by illuminating a user-defined area on the firstsubstrate. For example, a portion of the light-activated conductivematerial 3218 can be selected to correspond to a region of interest(ROI) 3222 on the sample 3212, and a beam of light 3224 is illuminatedonto the portion of the light-activated conductive material 3218 so thatthe portion of the light-activated conductive material 3218 beomesconductive and an electric field is generated between the portion of thelight-activated conductive material 3218 of the first substrate 3202 andthe second substrate 3204.

Although the first substrate 3202 is illustrated to include a singlesubstrate region 3216 in FIG. 4 , other implementations of the firstsubstrate 3202 can include a plurality of substrate regions that areconfigured to place multiple samples thereon, respectively. Each of suchmultiple substrate regions can be configured similarly to the firstsubstrate region 3216 described herein.

Referring still to FIG. 4 , the second substrate 3204 is configured tocomprise capture probes 3230. In some implementations, the secondsubstrate 3204 incudes a second substrate region 3232 configured toreceive the capture probes 3230. The second substrate 3204 is configuredto be used as a second electrode in the electrophoretic system 3200. Forexample, the second substrate 3204 can be used as an anode. In anotherexample, the second substrate 3204 can be used as a cathode. The captureprobe 3230 can be placed on the second substrate region 3232 in avariety of ways described herein. For example, the capture probes 3230can be directly or indirectly attached to a feature that is fixed on anarray. Alternatively or in addition, the capture probes 3230 can beimmobilized on the second substrate region 3232 of the second substrate3204.

The second substrate 3204 can be conductive at least in the secondsubstrate region 3232. In some implementations, the second substrate3204 can be configured as a conductive substrate described herein. Forexample, the second substrate 3204 can include one or more conductivematerials that permit the second substrate 3204 to function as anelectrode. Examples of such a conductive material include, but are notlimited to, tin oxide (TO), indium tin oxide (ITO), a transparentconductive oxide (TCO), aluminum doped zinc oxide (AZO), fluorine dopedtin oxide (FTO), and any combination thereof. Alternatively or inaddition, other materials may be used to provide desired conductivity tothe second substrate 3204. In some implementations, the second substrate3204 can be coated with the conductive material. For example, the secondsubstrate 3204 can include a conductive coating on the surface thereof,and the capture probes 3230 are provided on the coating of the secondsubstrate 3204. The second substrate can include a glass slide coatedwith a conductive material.

In some implementations, the second substrate 3204 can include an arrayof conductive regions that are selectively actuated, similarly to theconductive regions 3018 of the first substrate 3002 described in FIG. 1. In some implementations, the second substrate 3204 can have acustomized electrode pattern to improve capture uniformity. In otherimplementations, the second substrate 3204 can include a light-activatedconductive material similarly to the first substrate 3202.

In some implementations, the first substrate 3202 (or the firstsubstrate region 3216 thereof) and the second substrate 3204 (or thesecond substrate region 3232 thereof) can be flat and arranged inparallel with each other. For example, the first conductive surface areaof the first substrate 3202 and the second conductive surface area ofthe second substrate 3204 can be flat and arranged in parallel with eachother. Alternatively, at least one of the first substrate 3202 (or thefirst substrate region 3216 thereof, or the first conductive surfacearea thereof) and the second substrate 3204 (or the second substrateregion 3232 thereof, or the second conductive surface area thereof) isnot flat (e.g., curved). In addition or alternatively, the firstsubstrate 3202 (or the first substrate region 3216 thereof, or the firstconductive surface area thereof) can be arranged to be angled with thesecond substrate 3204 (or the second substrate region 3232 thereof, orthe second conductive surface area thereof).

In some implementations, the first substrate 3202 (or the firstsubstrate region 3216 thereof, or the first conductive surface areathereof) and the second substrate 3204 (or the second substrate region3232 thereof, or the second conductive surface area thereof) can beshaped to be concentric circular or spherical disks. Otherconfigurations and arrangements are also possible, such as square orrectangular shapes that are arranged concentrically ornon-concentrically.

Referring to FIG. 4 , the first substrate 3202 and the second substrate3204 can be arranged within the electrophoretic container 3206. Theelectrophoretic container 3206 can provide a buffer chamber 3250 betweenthe first substrate 3202 and the second substrate 3204. The bufferchamber 3250 is configured to contain a buffer 3252. In someimplementations, the first substrate 3202 and the second substrate 3204can be fully immersed into the buffer 3252. In alternativeimplementations, either or both of the substrates 3202, 3204 can bepartially inserted into the buffer 3252 contained in the electrophoreticcontainer 3206.

The buffer 3252 can be of various types. In some implementations, thebuffer 3252 includes a permeabilization reagent. The buffer 3252 iscontained in the buffer chamber 3250 throughout the electrophoreticprocess. The permeabilization reagent can permeabilize the sample beforeand/or during electrophoresis. In some implementations, this chemicalpermeabilization can be performed in conjunction with lightpermeabilization such as those described herein, for example withreference to FIG. 5 . In addition or alternatively, the sample can bepermeabilized using other methods described herein, independently or inconjunction with permeabilization using the premeabilization reagentand/or the illumination on the sample.

The spacer 3208 can be disposed between the first substrate 3202 and thesecond substrate 3204 to space them apart at a distance D. The spacer3208 is made of non-conductive material, such as plastic, glass,porcelain, rubber, silicone, etc. The distance D can be determined toprovide a desired level of spatial resolution based on several factors,such as the strength and/or duration of electric field generated betweenthe first substrate 3202 and the second substrate 3204, and otherparameters described herein.

The control system 3210 operates to generate an electric field (-E)between the first substrate 3202 and the second substrate 3204. Thecontrol system 3210 can include a controller 3260 configured to apply avoltage between the first substrate 3202 and the second substrate 3204using a power supply 3262. The power supply 3262 can include a highvoltage power supply. The controller 3260 can be electrically connectedto the first substrate 3202 and the second substrate 3204 usingelectrical wires. The control system 3210 can include a user interface3264 configured to receive a user input of starting or stopping theelectrophoretic process. The user interface 3264 can include varioustypes of input devices, such as physical or virtual buttons, GUI,switches, keypads, keyboard, etc., configured to receive a user input ofadjusting operating parameters of the system 3200 or other informationassociated with the system 3200. Examples of such operating parameterscan include a voltage being applied, a duration of such application,etc. In some implementations, the input devices can be used to select asubset of conductive regions 3218 that corresponds to a region ofinterest (ROI) 3222 on the sample 3212 so that the subset of conductiveregions are electrically activated to generate an electric field betweenthe subset of conductive regions 3218 and the second substrate 3204. Inaddition, the user interface 3264 can include an output device, such asa display, lamps, etc., configured to output the operating parameters ofthe system 3200 or other information associated with the system 3200.

The control system 3210 can further include a light generator 3266. Thelight generator 3266 is configured to generate and emit light on thefirst substrate 3202. Examples of light that can be used include anultraviolet (UV) light, visible light, or infrared light. In someimplementations, the light generator 3266 is manually controllable to bepositioned relative to the first substrate 3202, and also manuallycontrollable to generate and emit light to a particular area (e.g., theROI 3222) on the first substrate 3202. For example, the light generator3266 can be controlled based on a user input received through the userinterface 3264. Alternatively or in addition, the light generator 3266is configured to automatically adjust its position relative to the firstsubstrate 3202 and generate and emit light toward a particular area(e.g., the ROI 3222) on the first substrate 3202.

The controller 3260 can further control the light generator 3266. Insome implementations, the controller 3260 can control the lightgenerator 3266 to arrange or orient the light generator 3266 in adesired position relative to the substrate 3202, and/or generate andproject light onto the ROI 3222 so that a portion of the light-activatedconductive material 3218 corresponding to the ROI 3222 is activated tobe conductive.

Referring still to FIG. 4 , in some implementations, the first substrate3202 has a first conductive surface area that can be used as the firstelectrode in the electrophoretic system 3200, and the second substrate3204 has a second conductive surface area that can be used as the secondelectrode in the electrophoretic system 3200. The first conductivesurface area of the first substrate 3202 can be defined by the portionof the light-activated conductive material 3218 that is exposed to thelight beam 3224. Where the entire second substrate region 3232 isconfigured and used for the second electrode, the surface area of thesecond substrate region 3232 can be the second conductive surface area.Alternatively, where a portion of the second substrate region 3232 isconfigured and used for the second electrode, the surface area of theportion of the second substrate region 3232 can be the second conductivesurface area.

In some implementations, the first conductive surface area of the firstsubstrate 3202 (e.g., the portion of the light-activated conductivematerial 3218 being exposed to light) is different from the secondconductive surface area of the second substrate 3204, so that adivergent or convergent electric field is generated between the firstand second conductive surface areas in the electrophoretic system 3200.

As illustrated in FIG. 4 , a divergent electric field 3240 can begenerated when the first conductive surface area of the first substrate3202 (e.g., the portion of the light-activated conductive material 3218being exposed to light) is smaller than the second conductive surfacearea of the second substrate 3204. In the divergent electric field 3240,the analytes 3214 can migrate from the sample 3212 on the firstsubstrate 3202, toward the second substrate 3204, in a diverging manner.For example, the analytes 3214 are spread out during their travelthrough the divergent electric field 3240 and captured on the secondsubstrate 3204. In this case, the analytes 3214 captured on the secondsubstrate 3204 are considered to be magnified, compared to thoseoriginally included in the sample 3212 on the first substrate 3202.

In contrast, a convergent electric field can be generated when the firstconductive surface area of the first substrate 3202 (e.g., the portionof the light-activated conductive material 3218 being exposed to light)is larger than the second conductive surface area of the secondsubstrate 3204. In the convergent electric field, the analytes 3214 canmigrate from the sample 3212 on the first substrate 3202, toward thesecond substrate 3204 in a converging manner. For example, the analytes3214 can be concentrated while passing through the convergent electricfield 3242 and captured on the second substrate 3204. In this case, theanalytes 3214 captured on the second substrate 3204 are considered to bedemagnified, compared to those originally included in the sample 3212 onthe first substrate 3202.

Magnification or demagnification can be controlled by using differentsizes of the first electrode of the first substrate 3202 (e.g., theportion of the light-activated conductive material 3218 being exposed tolight) and the second electrode of the second substrate 3204. Forexample, magnification or demagnification can be adjusting based on aratio of the first conductive surface area of the first substrate 3202(e.g., the portion of the light-activated conductive material 3218 beingexposed to light) over the second conductive surface area of the secondsubstrate 3204. In some implementations, the ratio between the firstconductive surface area and the second conductive surface area can rangebetween 0.1 and 100. In other implementations, the ratio between thefirst conductive surface area and the second conductive surface area canrange between 0.01 and 1000. Other ratios are also possible.

In addition or alternatively, the distance D between the first substrate3202 and the second substrate 3204 can be controlled to adjust themagnification or demagnification. Further, the distance D can becontrolled to suppress low resolution capture of the analytes that mayresult from lateral diffusion. For example, the distance D can beselected to maintain a diffusion rate of the analytes migrating under adivergent or convergent electric field to be lower than a predeterminedvalue. In some implementations, the distance D can range from 1 um to 10mm. In other implementations, the distance D can range from 0.1 um to 10mm.

Referring to FIG. 5 , the electrophoretic system 3200 can furtherinclude a mechanism for providing photo-thermal effect to permeabilizethe sample before, during, or after operation of the electrophoreticsystem. For example, the control system 3210 includes a permeabilizer3268 configured to emit a beam of light 3270 (e.g., infrared light) ontothe sample placed on the first substrate 3202. In some implementations,the light 3270 can be emitted onto the same area (e.g., corresponding tothe ROI 3222) to which the light beam 3224 is emitted toward the portionof the light-activated conductive material 3218. The light beam 3270 canthermally activate permeabilizing enzymes in or in proximity to thesample locally in the ROI region, thereby enhancing migration of theanalytes toward the second substrate 3204.

FIG. 6 is a flowchart of an example process 3300. In someimplementations, the process 3300 can be performed using theelectrophoretic system 3000 described with reference to FIGS. 2 and 3 .The process 3300 can include placing a sample on a first substrate(3302), and placing a capture probe on a second substrate (3304). Thesample can be placed on the first substrate in various ways describedherein. The capture probe can be attached to the second substrate invarious ways described herein. As described herein, the first and secondsubstrates can be configured and used as electrophoretic electrodes. Insome implementations, the first and second substrates can be configuredas conductive substrates as described herein, such as by including aconductive material in the substrates or providing a conductive coatingon an upper or lower surface of the substrates.

As described herein, a conductive surface area of the first substrate isdifferent from a conductive surface area of the second substrate. Wherethe conductive surface area of the first substrate is smaller than theconductive surface area of the second substrate, a divergent electricfield is generated between the first and second substrates, and theanalytes can be captured on the second substrate in a magnified way.Where the conductive surface area of the first substrate is larger thanthe conductive surface area of the second substrate, a convergentelectric field is generated between the first and second substrates, theanalytes can be captured on the second substrate in a demagnified way.

The process 3300 can include providing a buffer between the firstsubstrate and the second substrate (3306). In some implementations, theprocess 3300 can include arranging a spacer between the first and secondsubstrates so that the first substrate is arranged at a distance fromthe second substrate. The distance can be selected to maintain adiffusion rate of the analytes migrating under the electric field to belower than a predetermined value. As described herein, the spacer can bemade of a non-conductive material and used to provide a buffer chamberbetween the first and second electrodes. The buffer can be contained ina buffer chamber that is provided by the spacer and used to at leastpartially immerse the first substrate, the second substrate, or both. Insome implementations, the buffer can include a permeabilization reagent.The permeabilization reagent can permeabilize the sample before and/orduring electrophoresis. In some implementations, this chemicalpermeabilization can be performed in conjunction with lightpermeabilization such as those described herein, for example withreference to FIG. 5 . In addition or alternatively, the sample can bepermeabilized using other methods described herein, independently or inconjunction with permeabilization using the premeabilization reagentand/or the illumination on the sample. Other buffers as describedgenerally herein can be used in other implementations.

The process 3300 can include identifying a region of interest (ROI) onthe first substrate (3308). The ROI can be selected to target a subsetof the sample that is of particular interest and capture the analytestherefrom. The process 3300 can include generating an electric field(e.g., a divergent or convergent electric field) between the ROI of thefirst substrate and the second substrate (3310). For example, the firstsubstrate can be selectively activated such that only the region of thefirst substrate corresponding to the ROI can be used as an electrode inthe electrophoresis. The electric field generated between the ROI of thefirst substrate and the second substrate can cause analytes to migratefrom the ROI of the first substrate toward the second substrate in adiverging or converging manner depending on the type of the electricfield (e.g., either a divergent electric field or a convergent electricfield). In some implementations, the first substrate includes alight-activated conductive film that is activated to be electricallyconductive when exposed to light of predetermined wavelength. Thelight-activated conductive film on the surface of the first substratecan be selectively activated to adjust the size of the first electrodein the electrophoretic system. The light-activated conductive film canbe used to create a free form of customized region of interest (ROI) byilluminating a user-defined area on the first substrate.

The process 3300 can include emitting light onto the ROI of the firstsubstrate to generate an electric field between the ROI of the firstsubstrate and the second substrate (3310). For example, the light can bea photoconductive light that is emitted onto the ROI of the firstsubstrate so that the region of the light-activated conductive filmcorresponding to the ROI is activated to be conductive. Thelight-emitted region (the ROI) has a size different from the secondsubstrate so that a divergent or convergent electric field is generatedbetween the first substrate and the second substrate.

The process 3300 can include emitting permeabilizing light onto thefirst substrate (3312). For example, the permeabilizing light (e.g.,infrared light) can be illuminated onto the area of the sample thatcorresponds to the portion of the light-activated conductive film ontowhich the light is emitted in the step 3310. The permeabilizing lightcan thermally permeabilize the sample locally, thereby enhancingmigration of the analytes toward the second substrate.

In some implementations, the permeabilizing light can have a wavelengthclose to infrared or in infrared (e.g., 1.5-2.0 um wavelengths). Thephotoconductive light can have a wavelength on UV or visible ranges.

FIG. 7 illustrates an example configuration of electrodes that can beused in the electrophoretic system described herein. In the illustratedexample, a first electrode 3402 is a source electrode, and a secondelectrode 3404 is a target or capture electrode.

The first electrode 3402 can represent the first substrate 3002, 3402.Alternatively, the first electrode 3402 can represent the firstsubstrate region 3016, 3216. Alternatively, the first electrode 3402 canrepresent the first conductive surface area of the first substrate 3002described herein. For example, the first electrode 3402 can represent aportion of the conductive regions 3018 of the first substrate 3002 thatis activated to be the first conductive surface area, as describedherein. Alternatively, the first electrode 3402 can represent a portionof the light-activated conductive material 3218 that is light-activatedto be conductive, as described herein.

The second electrode 3404 can represent the second substrate 3004, 3404.Alternatively, the second electrode 3404 can represent the secondsubstrate region 3032, 3232. Alternatively, the second electrode 3404can represent the second conductive surface area of the second substrate3004 described herein.

In some implementations, as illustrated in FIG. 7 , the first electrode3402 can provide a first spherical surface 3412, and the secondelectrode 3404 can provide a second spherical surface 3414. The firstelectrode 3402 and the second electrode 3404 can be disposedconcentrically so that an electric field 3420 is uniformly createdwithout distortion between any location of the first spherical surface3412 of the first electrode 3402 and its corresponding location of thesecond spherical surface 3414 of the second electrode 3404.

In other implementations, as illustrated in FIGS. 1, 2A-B, 4, and 5, thefirst electrode 3402 and the second electrode 3404 can be planar andarranged to be parallel with each other. The first electrode 3402 andthe second electrode 3404 can be of various shapes, such as circular,rectangular, square, or triangular disks or plates. In someimplementations, a potential electric field distortion that may resultfrom the shapes of the first and second electrodes can be used toreconstruct an undistorted profile of the captured analyte.

In yet other implementations, a combination of different shapes of thefirst electrode 3402 and the second electrode 3404 can be used.

In some implementations, the sizes (e.g., length or diameter) of thefirst and second electrodes 3402, 3404 can range from 1 um to 10 mm. Inother implementations, the sizes (e.g., length or diameter) of the firstand second electrodes 3402, 3404 can range from 5 um to 1 cm. In someimplementations, the distance between the first and second electrodes3402, 3404 can range from 1 um to 1 cm. In other implementations, thedistance between the first and second electrodes 3402, 3404 can rangefrom 0.1 um to 10 mm.

FIG. 8 is a schematic diagram showing an exemplary capture probe, asdescribed herein. As shown, the capture probe 102 is optionally coupledto a feature 101 by a cleavage domain 103, such as a disulfide linker.The capture probe can include a functional sequence 104 that are usefulfor subsequent processing. The functional sequence 104 can include allor a part of sequencer specific flow cell attachment sequence (e.g., aP5 or P7 sequence), all or a part of a sequencing primer sequence,(e.g., a R1 primer binding site, a R2 primer binding site), orcombinations thereof. The capture probe can also include a spatialbarcode 105. The capture probe can also include a unique molecularidentifier (UMI) sequence 106. While FIG. 8 shows the spatial barcode105 as being located upstream (5′) of UMI sequence 106, it is to beunderstood that capture probes wherein UMI sequence 106 is locatedupstream (5′) of the spatial barcode 105 is also suitable for use in anyof the methods described herein. The capture probe can also include acapture domain 107 to facilitate capture of a target analyte. In someembodiments, the capture probe comprises one or more additionalfunctional sequences that can be located, for example between thespatial barcode 105 and the UMI sequence 106, between the UMI sequence106 and the capture domain 107, or following the capture domain 107. Thecapture domain can have a sequence complementary to a sequence of anucleic acid analyte. The capture domain can have a sequencecomplementary to a connected probe described herein. The capture domaincan have a sequence complementary to a capture handle sequence presentin an analyte capture agent. The capture domain can have a sequencecomplementary to a splint oligonucleotide. Such splint oligonucleotide,in addition to having a sequence complementary to a capture domain of acapture probe, can have a sequence of a nucleic acid analyte, a sequencecomplementary to a portion of a connected probe described herein, and/ora capture handle sequence described herein.

The functional sequences can generally be selected for compatibilitywith any of a variety of different sequencing systems, e.g., Ion TorrentProton or PGM, Illumina sequencing instruments, PacBio, Oxford Nanopore,etc., and the requirements thereof. In some embodiments, functionalsequences can be selected for compatibility with non-commercializedsequencing systems. Examples of such sequencing systems and techniques,for which suitable functional sequences can be used, include (but arenot limited to) Ion Torrent Proton or PGM sequencing, Illuminasequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing.Further, in some embodiments, functional sequences can be selected forcompatibility with other sequencing systems, includingnon-commercialized sequencing systems.

In some embodiments, the spatial barcode 105 and functional sequences104 is common to all of the probes attached to a given feature. In someembodiments, the UMI sequence 106 of a capture probe attached to a givenfeature is different from the UMI sequence of a different capture probeattached to the given feature.

Spatial analysis methodologies and compositions described herein canprovide a vast amount of analyte and/or expression data for a variety ofanalytes within a biological sample at high spatial resolution, whileretaining native spatial context. Spatial analysis methods andcompositions can include, e.g., the use of a capture probe including aspatial barcode (e.g., a nucleic acid sequence that provides informationas to the location or position of an analyte within a cell or a tissuesample (e.g., mammalian cell or a mammalian tissue sample) and a capturedomain that is capable of binding to an analyte (e.g., a protein and/ora nucleic acid) produced by and/or present in a cell. Spatial analysismethods and compositions can also include the use of a capture probehaving a capture domain that captures an intermediate agent for indirectdetection of an analyte. For example, the intermediate agent can includea nucleic acid sequence (e.g., a barcode) associated with theintermediate agent. Detection of the intermediate agent is thereforeindicative of the analyte in the cell or tissue sample.

Non-limiting aspects of spatial analysis methodologies and compositionsare described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022,10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810,9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent ApplicationPublication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641,2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709,2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322,2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875,2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO2020/176788, Rodrigues et al., Science 363(6434):1463-1467, 2019; Lee etal., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gaoet al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol.36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits UserGuide (e.g., Rev C, dated June 2020), and/or the Visium Spatial TissueOptimization Reagent Kits User Guide (e.g., Rev C, dated July 2020),both of which are available at the 10× Genomics Support Documentationwebsite, and can be used herein in any combination. Further non-limitingaspects of spatial analysis methodologies and compositions are describedherein.

Some general terminology that may be used in this disclosure can befound in Section (I)(b) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Typically, a “barcode” is a label, oridentifier, that conveys or is capable of conveying information (e.g.,information about an analyte in a sample, a bead, and/or a captureprobe). A barcode can be part of an analyte, or independent of ananalyte. A barcode can be attached to an analyte. A particular barcodecan be unique relative to other barcodes. For the purpose of thisdisclosure, an “analyte” can include any biological substance,structure, moiety, or component to be analyzed. The term “target” cansimilarly refer to an analyte of interest.

Analytes can be broadly classified into one of two groups: nucleic acidanalytes, and non-nucleic acid analytes. Examples of non-nucleic acidanalytes include, but are not limited to, lipids, carbohydrates,peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins,phosphoproteins, specific phosphorylated or acetylated variants ofproteins, amidation variants of proteins, hydroxylation variants ofproteins, methylation variants of proteins, ubiquitylation variants ofproteins, sulfation variants of proteins, viral proteins (e.g., viralcapsid, viral envelope, viral coat, viral accessory, viralglycoproteins, viral spike, etc.), extracellular and intracellularproteins, antibodies, and antigen binding fragments. In someembodiments, the analyte(s) can be localized to subcellular location(s),including, for example, organelles, e.g., mitochondria, Golgi apparatus,endoplasmic reticulum, chloroplasts, endocytic vesicles, exocyticvesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) canbe peptides or proteins, including without limitation antibodies andenzymes. Additional examples of analytes can be found in Section (I)(c)of WO 2020/176788 and/or U.S. Patent Application Publication No.2020/0277663. In some embodiments, an analyte can be detectedindirectly, such as through detection of an intermediate agent, forexample, a ligation product or an analyte capture agent (e.g., anoligonucleotide-conjugated antibody), such as those described herein.

A “biological sample” is typically obtained from the subject foranalysis using any of a variety of techniques including, but not limitedto, biopsy, surgery, and laser capture microscopy (LCM), and generallyincludes cells and/or other biological material from the subject. Insome embodiments, a biological sample can be a tissue section. In someembodiments, a biological sample can be a fixed and/or stainedbiological sample (e.g., a fixed and/or stained tissue section).Non-limiting examples of stains include histological stains (e.g.,hematoxylin and/or eosin) and immunological stains (e.g., fluorescentstains). In some embodiments, a biological sample (e.g., a fixed and/orstained biological sample) can be imaged. Biological samples are alsodescribed in Section (I)(d) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, a biological sample is permeabilized with one ormore permeabilization reagents. For example, permeabilization of abiological sample can facilitate analyte capture. Exemplarypermeabilization agents and conditions are described in Section(I)(d)(ii)(13) or the Exemplary Embodiments Section of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

Array-based spatial analysis methods involve the transfer of one or moreanalytes from a biological sample to an array of features on asubstrate, where each feature is associated with a unique spatiallocation on the array. Subsequent analysis of the transferred analytesincludes determining the identity of the analytes and the spatiallocation of the analytes within the biological sample. The spatiallocation of an analyte within the biological sample is determined basedon the feature to which the analyte is bound (e.g., directly orindirectly) on the array, and the feature's relative spatial locationwithin the array.

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte (e.g., an analyte ofinterest) in a biological sample. In some embodiments, the capture probeis a nucleic acid or a polypeptide. In some embodiments, the captureprobe includes a barcode (e.g., a spatial barcode and/or a uniquemolecular identifier (UMI)) and a capture domain). In some embodiments,a capture probe can include a cleavage domain and/or a functional domain(e.g., a primer-binding site, such as for next-generation sequencing(NGS)). See, e.g., Section (II)(b) (e.g., subsections (i)-(vi)) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Generation of capture probes can be achieved by any appropriate method,including those described in Section (II)(d)(ii) of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

In some embodiments, more than one analyte type (e.g., nucleic acids andproteins) from a biological sample can be detected (e.g., simultaneouslyor sequentially) using any appropriate multiplexing technique, such asthose described in Section (IV) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, detection of one or more analytes (e.g., proteinanalytes) can be performed using one or more analyte capture agents. Asused herein, an “analyte capture agent” refers to an agent thatinteracts with an analyte (e.g., an analyte in a biological sample) andwith a capture probe (e.g., a capture probe attached to a substrate or afeature) to identify the analyte. In some embodiments, the analytecapture agent includes: (i) an analyte binding moiety (e.g., that bindsto an analyte), for example, an antibody or antigen-binding fragmentthereof; (ii) analyte binding moiety barcode; and (iii) an analytecapture sequence. As used herein, the term “analyte binding moietybarcode” refers to a barcode that is associated with or otherwiseidentifies the analyte binding moiety. As used herein, the term “analytecapture sequence” refers to a region or moiety configured to hybridizeto, bind to, couple to, or otherwise interact with a capture domain of acapture probe. In some cases, an analyte binding moiety barcode (orportion thereof) may be able to be removed (e.g., cleaved) from theanalyte capture agent. Additional description of analyte capture agentscan be found in Section (II)(b)(ix) of WO 2020/176788 and/or Section(II)(b)(viii) U.S. Patent Application Publication No. 2020/0277663.

There are at least two methods to associate a spatial barcode with oneor more neighboring cells, such that the spatial barcode identifies theone or more cells, and/or contents of the one or more cells, asassociated with a particular spatial location. One method is to promoteanalytes or analyte proxies (e.g., intermediate agents) out of a celland towards a spatially-barcoded array (e.g., includingspatially-barcoded capture probes). Another method is to cleavespatially-barcoded capture probes from an array and promote thespatially-barcoded capture probes towards and/or into or onto thebiological sample.

In some cases, capture probes may be configured to prime, replicate, andconsequently yield optionally barcoded extension products from atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent (e.g., a ligation product or an analyte captureagent), or a portion thereof), or derivatives thereof (see, e.g.,Section (II)(b)(vii) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663 regarding extended capture probes). In somecases, capture probes may be configured to form ligation products with atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent, or portion thereof), thereby creating ligationsproducts that serve as proxies for a template.

As used herein, an “extended capture probe” refers to a capture probehaving additional nucleotides added to the terminus (e.g., 3′ or 5′ end)of the capture probe thereby extending the overall length of the captureprobe. For example, an “extended 3′ end” indicates additionalnucleotides were added to the most 3′ nucleotide of the capture probe toextend the length of the capture probe, for example, by polymerizationreactions used to extend nucleic acid molecules including templatedpolymerization catalyzed by a polymerase (e.g., a DNA polymerase or areverse transcriptase). In some embodiments, extending the capture probeincludes adding to a 3′ end of a capture probe a nucleic acid sequencethat is complementary to a nucleic acid sequence of an analyte orintermediate agent specifically bound to the capture domain of thecapture probe. In some embodiments, the capture probe is extended usingreverse transcription. In some embodiments, the capture probe isextended using one or more DNA polymerases. The extended capture probesinclude the sequence of the capture probe and the sequence of thespatial barcode of the capture probe.

In some embodiments, extended capture probes are amplified (e.g., inbulk solution or on the array) to yield quantities that are sufficientfor downstream analysis, e.g., via DNA sequencing. In some embodiments,extended capture probes (e.g., DNA molecules) act as templates for anamplification reaction (e.g., a polymerase chain reaction).

Additional variants of spatial analysis methods, including in someembodiments, an imaging step, are described in Section (II)(a) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Analysis of captured analytes (and/or intermediate agents or portionsthereof), for example, including sample removal, extension of captureprobes, sequencing (e.g., of a cleaved extended capture probe and/or acDNA molecule complementary to an extended capture probe), sequencing onthe array (e.g., using, for example, in situ hybridization or in situligation approaches), temporal analysis, and/or proximity capture, isdescribed in Section (II)(g) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663. Some quality control measuresare described in Section (II)(h) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Spatial information can provide information of biological and/or medicalimportance. For example, the methods and compositions described hereincan allow for: identification of one or more biomarkers (e.g.,diagnostic, prognostic, and/or for determination of efficacy of atreatment) of a disease or disorder; identification of a candidate drugtarget for treatment of a disease or disorder; identification (e.g.,diagnosis) of a subject as having a disease or disorder; identificationof stage and/or prognosis of a disease or disorder in a subject;identification of a subject as having an increased likelihood ofdeveloping a disease or disorder; monitoring of progression of a diseaseor disorder in a subject; determination of efficacy of a treatment of adisease or disorder in a subject; identification of a patientsubpopulation for which a treatment is effective for a disease ordisorder; modification of a treatment of a subject with a disease ordisorder; selection of a subject for participation in a clinical trial;and/or selection of a treatment for a subject with a disease ordisorder.

Spatial information can provide information of biological importance.For example, the methods and compositions described herein can allowfor: identification of transcriptome and/or proteome expression profiles(e.g., in healthy and/or diseased tissue); identification of multipleanalyte types in close proximity (e.g., nearest neighbor analysis);determination of up- and/or down-regulated genes and/or proteins indiseased tissue; characterization of tumor microenvironments;characterization of tumor immune responses; characterization of cellstypes and their co-localization in tissue; and identification of geneticvariants within tissues (e.g., based on gene and/or protein expressionprofiles associated with specific disease or disorder biomarkers).

Typically, for spatial array-based methods, a substrate functions as asupport for direct or indirect attachment of capture probes to featuresof the array. A “feature” is an entity that acts as a support orrepository for various molecular entities used in spatial analysis. Insome embodiments, some or all of the features in an array arefunctionalized for analyte capture. Exemplary substrates are describedin Section (II)(c) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Exemplary features and geometricattributes of an array can be found in Sections (II)(d)(i),(II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Generally, analytes and/or intermediate agents (or portions thereof) canbe captured when contacting a biological sample with a substrateincluding capture probes (e.g., a substrate with capture probesembedded, spotted, printed, fabricated on the substrate, or a substratewith features (e.g., beads, wells) comprising capture probes). As usedherein, “contact,” “contacted,” and/or “contacting,” a biological samplewith a substrate refers to any contact (e.g., direct or indirect) suchthat capture probes can interact (e.g., bind covalently ornon-covalently (e.g., hybridize)) with analytes from the biologicalsample. Capture can be achieved actively (e.g., using electrophoresis)or passively (e.g., using diffusion). Analyte capture is furtherdescribed in Section (II)(e) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by attaching and/orintroducing a molecule (e.g., a peptide, a lipid, or a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) to a biologicalsample (e.g., to a cell in a biological sample). In some embodiments, aplurality of molecules (e.g., a plurality of nucleic acid molecules)having a plurality of barcodes (e.g., a plurality of spatial barcodes)are introduced to a biological sample (e.g., to a plurality of cells ina biological sample) for use in spatial analysis. In some embodiments,after attaching and/or introducing a molecule having a barcode to abiological sample, the biological sample can be physically separated(e.g., dissociated) into single cells or cell groups for analysis. Somesuch methods of spatial analysis are described in Section (III) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by detecting multipleoligonucleotides that hybridize to an analyte. In some instances, forexample, spatial analysis can be performed using RNA-templated ligation(RTL). Methods of RTL have been described previously. See, e.g., Credleet al., Nucleic Acids Res. 2017 Aug. 21; 45(14):e128. Typically, RTLincludes hybridization of two oligonucleotides to adjacent sequences onan analyte (e.g., an RNA molecule, such as an mRNA molecule). In someinstances, the oligonucleotides are DNA molecules. In some instances,one of the oligonucleotides includes at least two ribonucleic acid basesat the 3′ end and/or the other oligonucleotide includes a phosphorylatednucleotide at the 5′ end. In some instances, one of the twooligonucleotides includes a capture domain (e.g., a poly(A) sequence, anon-homopolymeric sequence). After hybridization to the analyte, aligase (e.g., SplintR ligase) ligates the two oligonucleotides together,creating a ligation product. In some instances, the two oligonucleotideshybridize to sequences that are not adjacent to one another. Forexample, hybridization of the two oligonucleotides creates a gap betweenthe hybridized oligonucleotides. In some instances, a polymerase (e.g.,a DNA polymerase) can extend one of the oligonucleotides prior toligation. After ligation, the ligation product is released from theanalyte. In some instances, the ligation product is released using anendonuclease (e.g., RNAse H). The released ligation product can then becaptured by capture probes (e.g., instead of direct capture of ananalyte) on an array, optionally amplified, and sequenced, thusdetermining the location and optionally the abundance of the analyte inthe biological sample.

During analysis of spatial information, sequence information for aspatial barcode associated with an analyte is obtained, and the sequenceinformation can be used to provide information about the spatialdistribution of the analyte in the biological sample. Various methodscan be used to obtain the spatial information. In some embodiments,specific capture probes and the analytes they capture are associatedwith specific locations in an array of features on a substrate. Forexample, specific spatial barcodes can be associated with specific arraylocations prior to array fabrication, and the sequences of the spatialbarcodes can be stored (e.g., in a database) along with specific arraylocation information, so that each spatial barcode uniquely maps to aparticular array location.

Alternatively, specific spatial barcodes can be deposited atpredetermined locations in an array of features during fabrication suchthat at each location, only one type of spatial barcode is present sothat spatial barcodes are uniquely associated with a single feature ofthe array. Where necessary, the arrays can be decoded using any of themethods described herein so that spatial barcodes are uniquelyassociated with array feature locations, and this mapping can be storedas described above.

When sequence information is obtained for capture probes and/or analytesduring analysis of spatial information, the locations of the captureprobes and/or analytes can be determined by referring to the storedinformation that uniquely associates each spatial barcode with an arrayfeature location. In this manner, specific capture probes and capturedanalytes are associated with specific locations in the array offeatures. Each array feature location represents a position relative toa coordinate reference point (e.g., an array location, a fiducialmarker) for the array. Accordingly, each feature location has an“address” or location in the coordinate space of the array.

Some exemplary spatial analysis workflows are described in the ExemplaryEmbodiments section of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. See, for example, the Exemplary embodimentstarting with “In some non-limiting examples of the workflows describedherein, the sample can be immersed . . . ” of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663. See also, e.g., theVisium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C,dated June 2020), and/or the Visium Spatial Tissue Optimization ReagentKits User Guide (e.g., Rev C, dated July 2020).

In some embodiments, spatial analysis can be performed using dedicatedhardware and/or software, such as any of the systems described inSections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663, or any of one or more of thedevices or methods described in Sections Control Slide for Imaging,Methods of Using Control Slides and Substrates for, Systems of UsingControl Slides and Substrates for Imaging, and/or Sample and ArrayAlignment Devices and Methods, Informational labels of WO 2020/123320.

Suitable systems for performing spatial analysis can include componentssuch as a chamber (e.g., a flow cell or sealable, fluid-tight chamber)for containing a biological sample. The biological sample can be mountedfor example, in a biological sample holder. One or more fluid chamberscan be connected to the chamber and/or the sample holder via fluidconduits, and fluids can be delivered into the chamber and/or sampleholder via fluidic pumps, vacuum sources, or other devices coupled tothe fluid conduits that create a pressure gradient to drive fluid flow.One or more valves can also be connected to fluid conduits to regulatethe flow of reagents from reservoirs to the chamber and/or sampleholder.

The systems can optionally include a control unit that includes one ormore electronic processors, an input interface, an output interface(such as a display), and a storage unit (e.g., a solid state storagemedium such as, but not limited to, a magnetic, optical, or other solidstate, persistent, writeable and/or re-writeable storage medium). Thecontrol unit can optionally be connected to one or more remote devicesvia a network. The control unit (and components thereof) can generallyperform any of the steps and functions described herein. Where thesystem is connected to a remote device, the remote device (or devices)can perform any of the steps or features described herein. The systemscan optionally include one or more detectors (e.g., CCD, CMOS) used tocapture images. The systems can also optionally include one or morelight sources (e.g., LED-based, diode-based, lasers) for illuminating asample, a substrate with features, analytes from a biological samplecaptured on a substrate, and various control and calibration media.

The systems can optionally include software instructions encoded and/orimplemented in one or more of tangible storage media and hardwarecomponents such as application specific integrated circuits. Thesoftware instructions, when executed by a control unit (and inparticular, an electronic processor) or an integrated circuit, can causethe control unit, integrated circuit, or other component executing thesoftware instructions to perform any of the method steps or functionsdescribed herein.

In some cases, the systems described herein can detect (e.g., registeran image) the biological sample on the array. Exemplary methods todetect the biological sample on an array are described in PCTApplication No. 2020/061064 and/or U.S. patent application Ser. No.16/951,854.

Prior to transferring analytes from the biological sample to the arrayof features on the substrate, the biological sample can be aligned withthe array. Alignment of a biological sample and an array of featuresincluding capture probes can facilitate spatial analysis, which can beused to detect differences in analyte presence and/or level withindifferent positions in the biological sample, for example, to generate athree-dimensional map of the analyte presence and/or level. Exemplarymethods to generate a two- and/or three-dimensional map of the analytepresence and/or level are described in PCT Application No. 2020/053655and spatial analysis methods are generally described in WO 2020/061108and/or U.S. patent application Ser. No. 16/951,864.

In some cases, a map of analyte presence and/or level can be aligned toan image of a biological sample using one or more fiducial markers,e.g., objects placed in the field of view of an imaging system whichappear in the image produced, as described in the Substrate AttributesSection, Control Slide for Imaging Section of WO 2020/123320, PCTApplication No. 2020/061066, and/or U.S. patent application Ser. No.16/951,843. Fiducial markers can be used as a point of reference ormeasurement scale for alignment (e.g., to align a sample and an array,to align two substrates, to determine a location of a sample or array ona substrate relative to a fiducial marker) and/or for quantitativemeasurements of sizes and/or distances.

1. An electrophoretic system for analyte migration, the systemcomprising: a first substrate including a first substrate regioncomprising a biological sample, and the first substrate regionconfigured to be electrically conductive and having a first surfacearea; a second substrate including a second substrate region configuredto receive analytes from the biological sample, the second substrateregion configured to be electrically conductive and having a secondsurface area; a buffer chamber comprising a buffer between the firstsubstrate region and the second substrate region; and a controllerconfigured to generate an electric field between the first substrateregion and the second substrate region such that the analytes migratefrom the first substrate region toward the second substrate region,wherein the first surface area of the first substrate region is largeror smaller than the second surface area of the second substrate region.2. An electrophoretic system for capturing an analyte from a biologicalsample, the system comprising: a first substrate including a firstsubstrate region, and the first substrate region including aphotoconductive material; a second substrate including a secondsubstrate region, the second substrate region configured to beelectrically conductive; a buffer chamber comprising a buffer betweenthe first substrate region and the second substrate region; a firstlight generator configured to emit a first light onto a least a portionof the first substrate region to permit for the at least a portion ofthe first substrate region to be electrically conductive; and acontroller configured to, based on the first light being emitted ontothe at least a portion of the first substrate region, generate anelectric field between the at least a portion of the first substrateregion and the second substrate region, wherein a surface area of the atleast a portion of the first substrate region is different from asurface area of the second substrate region.